Hoehnea 43(2): 289-300, 1 tab., 32 fig., 2016 http://dx.doi.org/10.1590/2236-8906-81/2015 Cultural and ligninolytic activity studies of some polypores () from brazilian Atlantic Forest, São Paulo State, Brazil

Viviana Motato-Vásquez1,2, Ricardo Matheus Pires1, Vera Maria Valle Vitali1 and Adriana de Mello Gugliotta1

Received: 27.10.2015; accepted: 25.04.2016

ABSTRACT - (Cultural and ligninolytic activity studies of some polypores (Basidiomycota) from brazilian Atlantic Forest, São Paulo State, Brazil). During a fungi survey in the brazilian Atlantic Forest in São Paulo, São Paulo State, Brazil, polypores growing on decaying wood were collected and fragments of the basidiomata were used to obtain isolates in pure culture. A total of 37 isolates were obtained and 23 species were identified. Cultural features of Bjerkandera atroalba, Henningsia brasiliensis, Rigidoporus undatus, and Steccherinum reniforme were described for the first time. A total of 31 isolates were evaluated in terms of their ability to oxidize the Remazol brilliant blue R dye (RBBR) and guaiacol. The results of these tests indicate that all isolated species were associated with white rot in the wood. Laccase and peroxidase activities were detected by drop tests using solutions of α-naphthol and pyrogallol. Production of cellulases and siderophores was detected with carboxymethylcellulose (CMC) and chrome azurol S (CAS) agar, respectively. Keywords: , laccase, Neotropical region, peroxidase, xylophilous fungi

RESUMO - (Estudos de culturas e atividade ligninolítica de alguns políporos (Basidiomycota) da Mata Atlântica brasileira, São Paulo, SP, Brasil). Durante um levantamento de fungos na Mata Atlântica brasileira do Estado de São Paulo, SP, Brasil, fungos poroides crescendo em madeira em decomposição foram coletados e fragmentos dos basidiomas foram utilizados para obter culturas puras. Um total de 37 isolados foram obtidos e identificados em 23 espécies. Características das culturas de Bjerkandera atroalba, Henningsia brasiliensis, Rigidoporus undatus e Steccherinum reniforme são descritas pela primeira vez. Um total de 31 isolados foram avaliados em termos da capacidade para oxidar os corantes Remazol brilliant blue R (RBBR) e guaiacol. Resultados destes testes indicaram que todas as espécies estudadas são associadas com produção de podridão branca na madeira. Atividades lacase e peroxidase foram detectadas pelo teste de gotas usando soluções de α-naphthol e pyrogallol. Produção de celulases e sideróforos foram detectadas com o ágar carboxymethylcellulose (CMC) e chrome azurol S (CAS), respectivamente. Palavras-chave: Agaricomycetes, fungos xilófilos, lacase, peroxidase, Região Neotropical

Introduction in vitro and (iii) mating system as a character of great importance that underlines the biology of each species. Morphology until the latter part of the last century After this work, an increasing number of studies were was the main tool used to characterize and to develop developed recording those features for each taxon in the the and putative phylogeny of different literature. Stalpers (1978) published a major work of fungi groups (Rajchenberg 2011). Agaricomycetes cultural characteristics of and Corticiales have been studied in culture for about 100 years, and Nakasone (1990) presented a cultural description and published works such as Nobles (1965) made of 277 wood-inhabiting basidiomycetes, Rajchenberg the first attempt to understand polypores on the basis (2011) present a review of the available knowledge on of their cultural features studied in vitro. Her work nuclear behavior of the mycelium, mating system and stressed the importance of (i) ability to degrade wood number of nuclei of the basidiospores within polypore (brown vs. white rot types) as a key element when genera, among others. considering generic delimitation and taxonomy, (ii) For decades, the division based on the presence or detailed macro- and microscopic features of cultures absence of extracellular oxidase in cultures proposed

1. Instituto de Botânica, Núcleo de Pesquisa em Micologia, Av. Miguel Stéfano 3687, 04301-902 São Paulo, SP, Brasil 2. Corresponding author: [email protected] 290 Hoehnea 43(2): 289-300, 2016 by Nobles (1965) to differentiate the fungi with Enzymatic test - All the enzymatic tests were different capacities to degrade wood, white-rot fungi, inoculated with a 5 mm diam. agar plug, always in which breakdown lignin and cellulose, and brown-rot triplicate. Plates were incubated at 25 °C in the dark fungi, which breakdown cellulose and hemicellulose, for 14 days. was recognized like a character that have diagnostic Remazol Brillant Blue - R (RBBR) decolorization significance to separate different species of wood- test: was detected by formation of an orange zone in inhabiting fungi (Lundell et al. 2010). A recent the culture medium containing 2% (w/v) MEA with phylogenetic study by Riley et al. (2014) indicates 0.05 % RBBR (Sigma) (Machado et al. 2005). that this simple dichotomy of white rot vs. brown rot does not properly reflect the diversity of mechanisms Phenol-oxidases production (Laccase s.s.) - was by which wood-rotting fungi obtain nutrition. This Detected by formation of a reddish zone in the culture study suggest that a more nuanced categorization medium containing 0.2% sugarcane bagasse powder, scheme is needed to describe wood decay by species 0.01% guaiacol (Sigma) and 1.6% agar (Nishida et al. that degrade all cell wall polymers. 1998). The drop test (Stalpers 1978) was carried out on Nevertheless, the study of features of fungal the same plate using: (a) α -naphthol solution to detect cultures isolated in axenic conditions and the qualitative Laccase (0.1 M in 96% ethanol) and (b) pyrogallol detection of enzymes causing the decomposition of solution to detect Lignin Peroxidase (equal parts of lignin is still valuable as an identification tool, and 4% hydrogen peroxide and 1% pyrogallol, freshly a first approach to describe the underlying biology prepared). The production of Laccase and Lignin and biochemistry of the fungi (Nakasone 1990). This Peroxidase was detected by formation of a purple approach is also very useful to select isolates with and a yellowish-brown zone in the culture medium, potential use in biotechnological, biotransformation respectively. and bioremediation processes (Machado et al. 2005). Cellulase activity test - Was detected by formation of In this way, this study aimed: (1) to expand the a whitish zone in the culture medium containing 25 ml knowledge about the biology of polypores presents of modified basal medium (per liter): 0.5 g ammonium in the Parque Estadual da Cantareira and Parque tartrate, 0.5 g monobasic potassium phosphate Estadual da Serra do Mar, São Paulo State, Brazil, with (KH PO ), 0.05 g magnesium sulfate (MgSO ), culture studies, and (2) to increase the knowledge of 2 4 4 0.01 g calcium chloride dehydrate (CaCl 2H O), the enzymatic activity of some brazilian fungi, which 2 2 0.01 g manganese sulfate (MnSO ), 0.05 g cooper have been poorly studied to date. 4 (II) sulfate pentahydrate (CuSO45H2O) (Kirk Materials and methods et al. 1978), 15 g agar, 0.01 g congo red and 1.0 g carboxymethylcellulose (CMC). Study of pure cultures - Specimens were collected from Detection of siderophores production by a modification August 2011 to February 2015 in the Parque Estadual of chrome azurol S (CAS) agar plate assay - CAS-blue da Cantareira (PEC), (23°32'36"S, 46º37'59"W) and agar was prepared follow the modification proposed by Parque Estadual da Serra do Mar (PESM) - Núcleo Milagres et al. (1999). The CAS reaction was detected Santa Virginia (23º24' to 23º17'S, 45º03' W), São by change the agar color from blue to orange. Paulo, Brazil. The species identification was based on macroscopic and microscopic characters of Results and Discussion basidiomata. The specimens were deposited in the herbarium SP. Cultures were obtained from fresh basidiomata and grown in Malt Extract Agar (MEA). Antrodiella semisupina (Berk. & M.A. Curtis) The species codes describing the cultures follow the Ryvarden, Prelim. Polyp. Fl. East Africa: 261 Species Code of Nobles (1965) with the modifications (1980). summarized by Nakasone (1990). The isolates were Description of culture in Stalpers (1978). preserved in sterile distilled water (Castellani 1967, Species code: 2.3.8.24.25.32.36.39.43.44.53.54.55.60. Burdsall & Dorworth 1994) and deposited in the Coleção de Culturas de Algas, Cianobactérias e Comments: the velvety to woolly mat and unusually Fungos (CCIBt) of the Instituto de Botânica, São downy, white to yellowish-brown, the dimitic hyphal Paulo, Brazil. system with generative hyphae clamped and skeletal Vásquez et al.: Cultural studies of polypores 291 hyphae in the aerial mat are features of this species Coriolopsis rigida (Berk & Mont.) Murrill, N. Amer. studied in culture, which is consistent with the Fl. 9:75 (1908). observations made by Stalpers (1978). Description of culture in Neves (1998). Cultures examined: BRASIL. São Paulo: SP, PEC, Species code: 2.3.4.8.32.36.37.39.41.42.54.55. 28-II-2012, V. Motato-Vásquez, M.C. Westphalen & A.C. Bolaños 225 (CCIBt 3871). Comments: the fast growth, the downy to felty mat and the presence of exudates hyaline to yellowish in most Bjerkandera atroalba (Rick.) Westphalen & colonies characterizes this species in culture. Although Tomšovský, Mycological Progress14(100): 3 (2015) the hyphal system of the basidioma is trimitic, in Figures 1-2, 9-14 culture it was only possible to find generative hyphae Macromorphological characters - Plates covered in with clamps and brown skeletal hyphae. These results differ from those presented by Neves (1998), who the 1st week. Advancing zone: regular, appressed, described the presence of skeletal and connective feathery, becoming finely cottony, white. Aerial hyphae in the culture. mycelium: initially appressed, villose, flocculent, white to cream. Submerged mycelium: white to Cultures examined: BRASIL. São Paulo: SP, PEC, cream. Odor: undetermined, weak to absent. Reverse: 29-XI-2011, V. Motato-Vásquez 42 (CCIBt 3876). whitening. Datronia caperata (Berk.) Ryvarden, Mycotaxon 23: Micromorphological characters - Advancing zone: 172 (1985) generative hyphae simple septate and clamped, Description of culture in Bakshi et al. (1969). hyaline, frequently branched, with smooth and thin to thick-walled, 2.0-5.0 µm diam. Aerial mycelium: Species code: 2.3.8.34.36.39.43.(44).54.60. formed by generative hyphae with clamps, smooth Comments: this species is characterized in culture by and thin-walled, very branched, 4.0-7.0 µm diam. white mat and presence of terminal and intercalary Skeletal hyphae not observed. Chlamydospores: chlamydospores. present in most colonies, intercalary and terminals, usually ellipsoid, rarely subglobose, with thin wall Cultures examined: BRASIL. São Paulo: São Paulo, 12.5-20.0 × 9.0-14.0 µm diam. PEC, 29-XI-2011, V. Motato-Vásquez 41 (CCIBt 3877). Species code: 2.3.4.34.36.40.41.53.54.60. Dichomitus setulosus (Henn.) Masuka & Ryvarden, Comments: Molecular analysis confirmed that Mycol. Res. 103(9): 1130 (1999) Tyromyces atroalbus (Rick) Rajchenb. belongs to Bjerkandera P. Karst and is separated from Tyromyces Description of culture in Rajchenberg (1983) as P. Karst, which is supported by the morphological Megasporoporia setulosa (Henn.) Rajchenb. features, such as the darkening of the basidiomata Species code: 2.3.4.8.32.36.38.40.43.44.50.54.60. when dried and distinctly monomitic hyphal system (Westphalen et al. 2015). This study presents the first Comments: this culture presents a dextrinoid reaction description of the culture of Bjerkandera atroalba in the skeletal hyphae, which is a feature that generally characterized by the formation of chlamydospores and have been used to identify species of this a monomitic hyphal system with generative hyphae (Ryvarden 2004). The same reaction was observed by with clamps. This features are shared with the culture Neves (1998) in cultures of D. cavernulosus (Berk.) of B. adusta (Willd.) P. Karst. (as Polyporus adustus Masuka and Ryvarden, both species morphologically related. Dextrinoid reaction is absent in cultures of Willd.), another species of the genus, described by D. hexagonoides (Speg.) Robledo & Rajchenb. like Nobles (1965). This results differ with descriptions observed by Stalpers (1978), and Rajchenberg (1983) of cultures of species of Tyromyces in which Stalpers also noted that D. setulosus and D. hexagonoides (1978) described the presence of skeletal hyphae. differ in the mat texture and the presence of distinct This results presents additional data that supports the submerged generative hyphae, characters that could be transfer of this species to Bjerkandera. useful to distinguish between both species. However, Cultures examined: BRASIL. São Paulo: SP, PEC, other biological and phylogenetic studies are needed 27-VI-2012, V. Motato-Vásquez, M.C. Westphalen & to assist in the delimitation of species of the genus A.C. Bolaños 248 (CCIBt 3974). Dichomitus D.A. Reid. 292 Hoehnea 43(2): 289-300, 2016

Cultures examined: BRASIL. São Paulo: SP, PEC, Stalpers (1978) and Neves (1998). However, Neves 7-III-2012, V. Motato-Vásquez 151 (CCIBt 3872). (1998) noted in the cultures that the generative hyphae are lengthened in the colony margin to form a globular Fomes fasciatus (Sw.) Cooke, Grevillea 14(69): 21 pseudoparenchyma, feature not observed in our culture. (1885) Cultures examined: BRASIL. São Paulo: SP, PESM, Description of culture in Neves (1998). 18-IV-2013, R.M. Pires 34 (CCIBt 4082). Species code: 2.3.7.(8).(11).21.22.24.25.34.35.39.4 Fomitella supina (Sw.:Fr.) Murr. Bull. Torrey Bot. 2.45.51.53 Club 32: 365 (1905) Comments: the culture studied is characterized by white to yellowish orange mat, becoming ochraceous. Description of culture in Neves (1998). Broadly, our results agree with those obtained by Species code: 2.3.8.32.36.38.50.54.

Figures 1-8. Microscopic features. 1-2. Bjerkandera atroalba. 1. Generative hyphae. 2. Chlamydospores. 3-4. Henningsia brasiliensis. 3. Generative hyphae. 4. Cuticular cells. 5-6. Rigidoporus undatus. 5. Generative hyphae. 6. Cystidia. 7-8. Steccherinum reniforme. 7. Generative hyphae and arthroconidia. 8. Cuticular cells. Scale bar = 5 µm. Vásquez et al.: Cultural studies of polypores 293

Comments: Fomitella supina is morphologically Species code: 2.6.8.11.34.37.39.45.46.50.54.57. similar in culture with species of Fomitopsis P. Karst., except by the production of white rot in the former Comments: the culture of this species is characterized and the production of chlamydospores in the latter in culture by white and then dark brown mat with (Stalpers 1978, Neves 1998). small floccose areas. Microscopically the presence of hyaline to yellowish generative hyphae, simple Cultures examined: BRASIL. São Paulo: SP, PESM, septate, hyaline to brown skeletal hyphae, globose to 17-IV-2013, R.M. Pires 28 (CCIBt 4081). ovoid chlamydospores and irregular cells forming a Fomitiporia maxonii Murrill, N. Amer. Fl. 9(1): 11 pseudoparenchyma are characteristic. (1907). Cultures examined: BRASIL. São Paulo: SP, PEC, Description of culture in Nobles (1965). 6-XII-2011, V. Motato-Vásquez 76 (CCIBt 3874).

Figures 9-20. Enzymatic tests. 9-14. Bjerkandera atroalba. 9. Mycelial growths during six weeks. 10. Guaiacol oxidation. 11. Drop test. 12. RBBR decolorization. 13. Cellulase production, non-detected. 14. Siderophores production. 15-20. Henningsia brasiliensis. 15. Mycelial growths during six weeks. 16. Guaiacol oxidation. 17. Drop test. 18. RBBR decolorization. 19. Cellulase production, non-detected. 20. Siderophores production, non-detected. Plate = 9 cm. 294 Hoehnea 43(2): 289-300, 2016

Ganoderma australe (Fr.) Pat., Bull. Soc. Mycol. Inonotus tropicalis (M.J. Larsen & Lombard) T. France 5: 71 (1889). Wagner & M. Fisch., Mycologia 94(6): 1009 (2002) Description of culture in Bettucci & Guerrero (1971). Description of culture in Larsen & Lombard (1988) Species code: 2.3.8.10.32.37.39.(40).46.47.50.54.55. Species code: 2.6.7.(17).32.37.39.43.54. Comments: this species is very variable in culture, feature observed in the three different isolates obtained Comments: the moderately rapid growth on MEA, from different basidiomata. But it usually can be the yellow mat, often pale brown generative hyphae, recognized by the slow growth, cracking the agar simple-septate, dark brown skeletal hyphae, rare medium and wrinkling the colony surface. Besides of or absent, brown and narrowly subulate setae are the mat white and the abundant sphaerical cuticular characteristics of this species in culture, which are cells, features that were also observed by Bettucci & consistent with the observations carried out by Larsen Guerrero (1971) and Yeh et al. (2000). & Lombard (1988).

Cultures examined: BRASIL. São Paulo: SP, PEC, Cultures examined: BRASIL. São Paulo: SP, PESM, 6-XII-2011, V. Motato-Vásquez 72 (CCIBt 3972), 12-VI-2013, R.M. Pires 73 (CCIBt 4130). 28‑II-2012, V. Motato-Vásquez 124 (CCIBt 3973); PESM, 11-VI-2013, R.M. Pires 57 (CCIBt 4086). Junghuhnia nitida (Pers.) Ryvarden, Persoonia 7(1): 18 (1972) Henningsia brasiliensis (Speg.) Speg., Bol. Acad. Description of culture in Stalpers (1978). Nal. Cienc. Córdoba 23(3-4): 411 (1919). Figures 3-4, 15-20 Species code: 2.3.8.9.(11).13.(14).24.25.32.36. (38).39.42.52.54.57. Macromorphological characters - Plates covered in 5-6th week. Advancing zone: smooth, appressed, Comments: the cultural features of our specimen subfelty, white. Margin uneven. Aerial mycelium: are quite similar to those found by Stalpers (1978). cottony to woolly and later downy, initially white The white mat with salmon pores after 3-5 weeks, and later forming an orange to reddish-orange encrusted skeletal hyphae and variably present cystidia crust. Submerged mycelium: white to cream. Odor: sometimes present, apically somewhat swollen are undetermined. Reverse: unchanged. characteristics of this species in culture. Micromorphological characters - Advancing zone: Cultures examined: BRASIL. São Paulo: SP, PESM, generative hyphae simple septate, hyaline, straight, 12-VI-2013, R.M. Pires 79 (CCIBt 4092). branched, with smooth and thin-walled, 1.5-3.0 µm Oxyporus latemarginatus (Durieu & Mont.) Donk, diam. Aerial mycelium: as in the advancing zone. Persoonia 4(3): 342 (1966) Pseudoparenchyma: hyphae differentiated, with thickened walls presents in the crusty areas. Submerged Description of culture in Lombard et al. (1960). mycelium: as in the advancing zone. Species code: 2.4.7.10.35.36.38.41.50.54.55. Species code: 2.6.10.32.36.(37).38.45.46.53.54. Comments: the rapid growth on MEA, absence Comments: Henningsia brasiliensis has been closely of clamps, generative hyphae often encrusted and associated with the genus Rigidoporus Murrill absence of cystidia are characteristics of this species with regard to their morphological characteristics. in culture. The results presented here agree with those In culture, it is possible to relate the two genera reported by Lombard et al. (1960). by the coloration of the mat and the formation of Cultures examined: BRASIL. São Paulo: SP, PEC, pseudoparenchyma. But in the culture, H. brasiliensis 7-XI-2012, V. Motato-Vásquez et al. 283 (CCIBt could be separated microscopically by the absence 3975). of cystidia, when compared with the two species of Rigidoporus studied here: R. lineatus (Pers.) Ryv. and Polyporus tenuiculus (P. Beauv.) Fr., Syst. Mycol. R. microporus (Fr.) Overeem). 1: 344 (1821) Description of culture in Neves (1998). Cultures examined: BRASIL. São Paulo: SP, PEC, 7-XI-2012, V. Motato-Vásquez et al. 280 (CCIBt 3976). Species code: 2.3.8.35.36.38.45.46.53.54.60. Vásquez et al.: Cultural studies of polypores 295

Comments: the cottony and white mat, the dimitic white. Margin uneven. Aerial mycelium: initially hyphal system with clamped generative hyphae, white, later pinkish-salmon, appressed, and velvety to branched skeletal hyphae and abundant arthroconidia downy. Submerged mycelium: white to cream. Odor: characterizes the culture of this species. Neves (1998) acid. Reverse: yellowish-brown. observed the presence of crystals in the cultures Micromorphological characters: Advancing zone: studied, this results differ from our observations and generative hyphae simple septate, hyaline, straight, largely with those made by Stalpers (1978). branched, with smooth and thin wall, 2.0-4.5 µm Cultures examined: BRASIL. São Paulo: SP, PEC, diam. Aerial mycelium: as the advancing zone. 24-VIII-2012, V. Motato-Vásquez & M.C. Westphalen Pseudoparenchyma: differentiated hyphae with 275 (CCIBt 3963). thickened walls present in the crusty areas. Cystidia: clavate, spiny or smooth, 15.5−22.5 × 7.0−17.5 µm. Pycnoporus sanguineus (L.) Murrill, Bull. Torrey Submerged mycelium: as the advancing zone. Odor: Bot. Club 31(8): 421 (1904) like iodoform. Reverse: unchanged. Description of culture in Nobles (1965). Species code: 2.6.10.13.32.36.39.46.47.52.54.55. Species code: 2.3.8.34.35.36.38.41.42.53.54.55.60. Comments: comparing the culture of R. undatus with another cultures of species of the genus, they share Comments: the strong orange to pinkish-salmon the presence of simple septate generative hyphae, color of the culture is similar to the color of the single cells differentiated in the pseudoparenchyma basidioma. This is a very important characteristic and presence of cystidia. The differentiation between for easy recognition of the species in culture. Our the species of Rigidoporus in culture is difficult, results agree with those presented by Nobles (1965), but in this study we can separate R. lineatus from Bettucci & Guerrero (1971) and Wright & Deschamps R. undatus based on growth rate (2-4 weeks in the (1975). Neves (1998) observed the formation of former and 6-7 weeks in the latter) and the size of chlamydospores and rhomboid crystals near the mat cystidia (13.5‑18.5 × 6.0‑14.5 µm in the former and surface, characteristics absent in the cultures studied 15.5‑22.5 × 7.0‑17.5 µm in the latter). in this work. Cultures examined: BRASIL. São Paulo: SP, PEC, Cultures examined: BRASIL. São Paulo: SP, PEC, 29-XI-2011, V. Motato-Vásquez 38 (CCIBt 3958). 30-VII-2012, V. Motato-Vásquez & M.C. Westphalen Schizopora flavipora (Berk. & M.A. Curtis ex Cooke) 260 (CCIBt 3962); PESM, 7-VIII-2013, R.M. Pires Ryvarden, Mycotaxon 23: 186 (1985) 71 (CCIBt 4090). Description of culture in Stalpers (1978). Rigidoporus lineatus (Pers.) Ryvarden, Norw. J. Bot. Species code: 1.3.32.36.40.43.44.53.54.55.60. 19: 236 (1972) Comments: this species is characterized in culture Description of culture in Wright & Deschamps (1975). by white and thin mat, initially farinaceous to villose Species code: 2.6.10.13.32.36.39.43.44.51.54.55. and microscopically by the generative hypha with abundant clamps. Comments: the initially white and then orange mat, simple septate generative hyphae, pseudoparenchyma Cultures examined: BRASIL. São Paulo: SP, PEC, formed by swollen generative hyphae, the spiny or 27-VII-2012, V. Motato-Vásquez, M.C. Westphalen & smooth and clavate cystidia, (13.5‑18.5 × 6.0‑14.5 µm) A.C. Bolaños 256 (CCIBt 3965). are characteristics of this species. Steccherinum reniforme (Berk. & M.A. Curtis) Cultures examined: BRASIL. São Paulo: SP, PEC, Banker, Mem. Torrey Bot. Club 12: 127 (1906) 29-XI-2011, V. Motato-Vásquez 47 (CCIBt 3959). Figures 7-8, 27-32 Macromorphological characters: plates covered in Rigidoporus undatus (Pers.) Donk, Persoonia 5(1): the 1st week. Advancing zone: smooth, appressed, 115 (1967). cottony, white. Margin: uneven. Aerial mycelium: Figures 5-6, 21-26 appressed, cottony to downy, white to yellowish. Macromorphological characters: plates covered in Submerged mycelium: white to cream. Odor: 5-6th week. Advancing zone: regular, smooth, and undetermined. Reverse: bleached. 296 Hoehnea 43(2): 289-300, 2016

Micromorphological characters: Advancing zone: arthroconidia hyaline, smooth and with thick wall are generative hyphae with clamps, hyaline, straight, smooth, characteristics of this species. thin-walled, 1.5-2.5 µm diam. Aerial mycelium: as in the Cultures examined: BRASIL. São Paulo: SP, PEC, advancing zone. Arthroconidia: hyaline, smooth, thick- 30-VIII-2012, V. Motato-Vásquez & M.C. Westphalen walled. Submerged mycelium: as in the advancing zone 263 (CCIBt 3971). and hyphae differentiated through, hooked, thick-walled, and interlocked to form plectenchyma. Tinctoporellus empimiltinus (Berk. & Broome) Species code: 2.3.11.35.36.40.41.53.54. Ryvarden, Trans. Br. Mycol. Soc. 73: 19 (1979) Comments: colonies growing very fast, mat white Description of culture in Rajchenberg (1983). to yellowish, cottony, clamped generative hyphae, Species code: 2.4.8.10.11.34.35.37.38.(39).41.50.54.

Figures 21-32. Enzymatic tests. 21-26. Rigidoporus undatus. 21. Mycelial growths during six weeks. 22. Guaiacol oxidation. 23. Drop test. 24. RBBR decolorization. 25. Cellulase production. 26. Siderophores production. 27-32. Steccherinum reniforme. 27. Mycelial growths during six weeks. 28. Guaiacol oxidation. 29. Drop test. 30. RBBR decolorization, non-detected. 31. Cellulase production. 32. Siderophores production. Plate = 9 cm. Vásquez et al.: Cultural studies of polypores 297

Comments: Tinctoporellus epimiltinus showed chlamydospores was absent, different to the culture similar growth and microscopic characteristics of of T. elegans. Grammothele subargentea (Speg.) Rajchenb. in Cultures examined: BRASIL. São Paulo: SP, PEC, culture medium (Rajchenberg 1983). Both change the 20-IX-2011, V. Motato-Vásquez & A.M. Gugliotta 8, color of the culture medium, making it reddish and 9 (CCIBt 3966, CCIBt 3967), 29-XI-2011, V. Motato- form a plectenchyma. The basidioma of both species Vásquez 48 (CCIBt 3968), 8-V-2012, V. Motato- exhibit a similar hyphal system, with dextrinoid Vásquez 204 (CCIBt 3969); PESM, 11-VI-2013, R.M. skeletal hyphae and the production of white rot in Pires 55 (CCIBt 4084), 12-VI-2013, R.M. Pires 74 the substrate. In addition, different species of the (CCIBt 4091), 13-VI-2013, R.M. Pires 88 (CCIBt genus Grammothele Berk. & M.A. Curtis have also 4093), 8-VIII-2013, R.M. Pires 118 (CCIBt 4133). been transferred or synonymized with Porogramme (Pat.) Pat. species (see Soares et al. 2015), which also Trichaptum sector (Ehrenb.) Kreisel, Monogr. Cienc. indicates the narrow morphological and ecological Univ. Habana 16: 84 (1971) relationship of these three genus and the need for Description of culture in Alexander et al. (1989). taxonomic and phylogenetic revision with more specialized techniques like cytological and sexual Species code: 2.3.7.32.36.38.43.54.60.61. studies as commented by Rajchenberg (1983). Comments: colonies growing moderately fast, white and subfelty mat, clamped generative hyphae, and Cultures examined: BRASIL. São Paulo: SP, PEC, intercalary or terminal arthroconidia are characteristics 29-XI-2011, V. Motato-Vásquez 46 (CCIBt 3875), of this species. 28-II-2012, V. Motato-Vásquez 122 (CCIBt 3873). Cultures examined: BRASIL. São Paulo: SP, PEMS, Trametes elegans (Spreng.) Fr., Epicr. Syst. Mycol: 11-VI-2013, R.M. Pires 63, 67 (CCIBt 4088, CCIBt 491 (1838) 4129). Description of culture in Nobles (1965). Enzymatic test – A total of 37 specimens were Species code: 2.3.4.8.34.35.36.40.41.(42).50.54. isolated from basidiomata growing in decaying wood and classified into 23 species. Of all the isolated Comments: the culture of this species was examined obtained, 31 were qualitatively evaluated in terms and it was found to differ from the cultures of of their ligninolytic activity (table 1). All species another species of Trametes Fr. that were examined were associated with production of a white rot with by Stalper (1978) and Neves (1998) and also in this regard to the presence of Phenol-oxidases that was work, principally by the presence of chlamydospores confirmed for almost all specimens tested. In this test and arthroconidia besides of the generative hyphae only had negative results: A. semisupina (RBBR), simple-septate in the advance zone. S. flavipora (all tests), I. tropicalis (Guaiacol), S. Cultures examined: BRASIL. São Paulo: SP, PEC, reniforme (Laccase and RBBR) T. sector (Guaiacol) 6-XII-2011, V. Motato-Vásquez 67 (CCIBt 3960), and T. atroalbus (laccase, peroxidase and RBBR). 28-II-2012, V. Motato-Vásquez 131 (CCIBt 3961), These species were also associate with production 27-VI-2012, V. Motato-Vásquez, M.C. Westphalen & of a white rot from data obtained in the literature A.C. Bolaños 243 (CCIBt 3964). (Stalpers 1978, Larsen & Lombard 1988, Alexander et al. 1989). Currently it is known that negative results Trametes villosa (Sw.) Kreisel, Monogr. Cienc. Univ. in this tests may be related to the lack of specificity of Habana 16: 83 (1971) the enzymes in the oxidation of substrates tested or Description of culture in Susin (1989). absence of expression of the total ligninolytic system due to the culture conditions used (Platt et al. 1985). Species code: 2.3.8.32.36.40.41.42.50.54.55.60. Production of cellulolytic enzymes was detected Comments: the results obtained in this study are in 18 of the 31 isolates tested. In all the isolates the similar to those obtained by Susin (1989) and Neves growth was very poor. These results agree with the (1998), except that our culture not present connective literature, since the expression of the total cellulolytic hyphae and the reverse was not bleaching. The activity could differ due to the culture conditions generative hyphae in the margin presents clamps and (Arantes & Milagres 2009). 298 Hoehnea 43(2): 289-300, 2016

Detection of siderophores in CAS-agar occurred compounds (Gomes et al. 2009, Lyra et al. 2009). Other only in 18 of the 31 isolates tested and they differed less commonly used species such as Fomes fasciatus, F. in terms of growth and change of CAS-agar color. It supina, O. latemarginatus and R. undatus also performed is believed that fungi that produced low molecular well and showed very fast growth, features that make weight compounds are responsible for the initial them interesting for studies in biotechnological or process of degradation of wood (Arantes et al. 2011). bioremediation processes in laboratory scale. The results It should be noted that some of the isolates showing presented in this work suggested that these tests could positive results in all the biochemical tests (C. rigida, P. be used as a simple and effective way to indirectly select sanguineus, T. elegans and T. epimiltinus) present records fungi producers of lignocellulolytic enzymes, as an in the current literature being used in processes such important basis for future biotechnological, ecological as degradation of effluents and recalcitrant xenobiotic and molecular studies.

Table 1. Ligninolytic activity of polypores isolated from decaying wood. Production of enzymes: Phenol-Oxidase in Guaiacol (G) and RBBR test, production of Laccase (L), Peroxidase (P), Cellulase (C) and Siderophores (S). Detection of enzymes: positive (+) or negative (−).

Species CCIBt G RBBR L P C S A. semisupina 3871 + − + + + − B. atroalba 3974 + + − − − + C. rigida 3876 + + + + + + D. caperata 3877 + + + + − − D. setulosus 3872 + + + + + − F. fasciatus 4082 + + + + + + F. supina 4081 + + + + + + F. maxonii 3874 + + + + + − G. australe 3972 + + + + − + G. australe 3973 + + + + − + H. brasiliensis 3976 + + + + − − I. tropicalis 4130 − + + + + − J. nitida 4092 + + + + − − O. latemarginatus 3975 + + + + + + P. tenuiculus 3963 + + + + − − P. sanguineus 3962 + + + + + + R. lineatus 3959 + + + + + − R. undatus 3958 + + + + + + S. flavipora 3965 − − − − − − S. reniforme 3971 + − + − + + T. epimiltinus 3873 + + + + + + T. epimiltinus 3875 + + + + + + T. elegans 3960 + + + + + + T. elegans 3961 + + + + + + T. elegans 3964 + + + + − + T. villosa 3966 + + + + − + T. villosa 3967 + + + + − + T. villosa 3968 + + + + − + T. villosa 3969 + + + + − + T. sector 4088 − + + + + – T. sector 4092 − + + + + – Vásquez et al.: Cultural studies of polypores 299

Acknowledgements Lombard, F.F., Davidson, R.W. & Lowe, J.L. 1960. Cultural characteristics of Fomes ulmarius and Poria The author V. Motato-Vásquez gratefully ambigua. Mycologia 52: 280-296. acknowledges the financial support received from Lundell, T.K., Mäkelä, M.R. & Hildén, K. 2010. Lignin- the Programa Estudantes-Convênio Pós-Graduação - modifying enzymes in filamentous basidiomycetes - PEC-PG, from CAPES/CNPq - Brazil and the support ecological, functional and phylogenetic review. Journal received from Idea Wild. Instituto Florestal for collect of Basic Microbiology 50: 5-20. Lyra, F.S., Moreira, K.A., Porto, T.S., Carneiro da authorizations. RMP is grateful to the financial support Cunha, M.N., Paz Junior, F.B., Neto, B.B., Lima- received from Fundação de Amparo à Pesquisa do Filho, J.L., Cavalcanti, M.A.Q. & Porto, A.L.P. 2009. Estado de São Paulo - FAPESP (12/25493-7). Dra. Decolorization of synthetic dyes by basidiomycetes Karen Nakasone (U.S. Forest Service, USA) is warmly isolated from woods of the Atlantic Forest (PE), Brazil. acknowledged by donation of bibliographical material. World J Microbiol Biotechnol 25: 1499-1504. Machado, K.M.G., Matheus, D.R. & Bononi, V.L.R. Literature cited 2005. Ligninolytic enzymes production and Remazol Brilliant Blue R decolorization by tropical Brazilian Alexander, J., Welden, A.L., & Ovebro, C.L. 1989. basidiomycetes fungi. Brazilian Journal of Microbiology Trichaptum sector (Ehrenb.:Fr.) Kreisel ( 36: 246-252. s.l.). Mycologia Helvetica 3: 291-302. Milagres, M.F., Machuca, A. & Napoleão, A. 1999. Arantes, V. & Milagres, M.F. 2009. Relevância de Detection of siderophores production from several fungi and bacteria by a modification of chrome azurol compostos de baixa massa molar produzidos por fungos S (CAS) agar plate assay. Journal of Microbiological e envolvidos na degradação da madeira. Química Nova Methods 37: 1-6. 32: 1586-1595. Nakasone, K.K. 1990. Cultural studies and identification Arantes, V., Milagres, M.F., Timothy, R.F. & Goodell, of wood inhabiting Corticiaceae and selected B. 2011. Lignocellulosic polysaccharides and lignin Hymenomycetes from North America. Mycological degradation by wood decay fungi: the relevance of Memoirs 165: 1-412. nonenzymatic Fenton-based reactions. Journal Indian Neves, M.A. 1998. Estudos de culturas de fungos Microbiology and Biotechnology 38: 541-555. (Aphyllophorales) da Ilha de Santa Catarina, SC, Brasil. Bakshi, B.K., Sehgal, H.S. & Singh, B. 1969. Cultural Biotemas 11: 39-70. diagnosis of Indian Polyporaceae. I. Genus Polyporus. Nishida, T., Yoshinori, K., Mimura, A. & Takahara, Y. Indian Forest Research 2: 205-244. 1988. Lignin biodegradation by wood-rotting fungi Bettucci, L. & Guerrero, R. 1971. Hongos xilófagos: I. Screening of lignin degradation fungi. Mokuzai estudios de cultivo. Boletín de la Universidad de Gakkaishi 34: 530-536. Nobles, M.K. 1965. Identification of cultures of wood- la República, Facultad de Agronomía, Montevideo inhabiting Hymenomycetes. Canadian Journal of 118: 1-40. Botany 43: 1097-1139. Burdsall, H.H. & Dorworth, E.B. 1994. Preserving Platt, M.W., Hadar, Y. & Chet, I. 1985. The decolorization cultures of wood-decaying Basidiomycotina using of the polimeric dye poly-blue (polyvinilalamine sterile water in Cryovials. Mycologia 86: 275-280. sulfonate-anthroquinone) by lignin degrading fungi. Castellani, A. 1967. Maintenance and cultivation of Applied Microbiology and Biotechnology 21: 394-396. common pathogenic fungi in distilled water. The Journal Rajchenberg, M. 1983. Cultural studies of resupinate of Tropical Medicine and Hygiene 42: 181-184. polypores. Mycotaxon 17: 275-293. Gomes, E., Aguiar, A.P., Carvalho C.C., Bonfá, M.R.B. Rajchenberg, M. 2011. Nuclear behavior of the myceliumand & Boscolo, M. 2009. Ligninases production by the phylogeny of Polypores (Basidiomycota). Mycologia basidiomycetes strains on lignocellulosic agricultural 103: 677-702. residues and their application in the decolorization Riley, R., Salamov, A.A., Brown, D.W., Nagy, L.G., of synthetic dyes. Brazilian Journal of Microbiology Floudas, D., Held, B.W., Levasseur, A., Lombard, 40: 31‑39. V., Morin, E., Otillar, R., Lindquist, E.A., Sun, H., Labutti, K.M., Schmutz, J., Jabbour, D., Luo, Kirk, T.K., Schultz, E., Connors, W.J., Lorenz, L.F. & H., Baker, S.E., Pisabarro, A.G., Walton, J.D., Zeikus, J.G. 1978. Influence of culture parameters on Blanchette, R.A., Henrissat, B., Martin, F., Cullen, lignin metabolism by Phanerochaete chrysosporium. D., Hibbett, D.S. & Grigoriev, I.V. 2014. Extensive Archives of Microbiology 117: 277-285. sampling of basidiomycete genomes demonstrates Larsen, M.J. & Lombard, F.F. 1988. Studies in the genus inadequacy of the white-rot/brown-rot paradigm Phellinus. I. The identity of Phellinus rickii with notes for wood decay fungi. Proceedings of the National on its facultative synonyms. Mycologia 80: 72-76. Academy Sciences 111: 9923-9928. 300 Hoehnea 43(2): 289-300, 2016

Ryvarden, L. 2004. Neotropical Polypores. Part 1. Susin, V.L.N. 1989. Biología de Aphyllophorales xilófilas Introduction, Ganodermataceae and Hymenochaetaceae. (Basidiomycetes). Ínsula19: 57-76. Synopsis Fungorum 19: 1-227. Westphalen, M.C., Tomšovský, M., Jout, J. & Gugliotta, Soares, A.M.S., Gibertoni, T.B., Gomes-Silva, A.C., A.M. 2015. Bjerkandera in the Neotropics: phylogenetic Medeiros, P.S., Sotão, H.M.P. & Ryvarden, L. 2015. and morphological relations of Tyromyces atroalbus and description of a new species. Mycol Progress 14: 100-108. Validation of Porogramme albocincta and new records Wright, J.E. & Deschamps, J.R. 1975. Basidiomicetos of Polyporales from the Brazilian Amazonia. Journal of xilófilos de la región mesopotámica II. Los géneros the Torrey Botanical Society 142: 331-336. Daedalea, Fomitopsis, Heteroporus, Laetiporus, Stalpers, J.A. 1978. Identification of wood-inhabiting Nigroporus, Rigidoporus, y Vanderbylia. Aphyllophorales in pure culture. Studies in mycology Revista de Investigaciones Agropecuarias 12: 127-204. 16: 1-248. Available in http://www.cbs.knaw.nl/ Yeh, Z.Y., Chen, Z.C. & J.W. Kimbrough. 2000. publications/1016/full%20text.htm (access in Ganoderma australe from Florida. Mycotaxon 10‑II‑2015). 75: 233‑240.